POS0481 DEFINING DISTINCT RESIDENT AND MIGRATORY FIBROBLAST POPULATIONS FROM SKIN BIOPSIES IN SYSTEMIC SCLEROSIS
Authors: K. Clark, A. Cole, X. Shiwen, V. Ong, C. D. Buckley, C. P. Denton
Journal: Annals of the Rheumatic Diseases (2022)

Background Recent studies using single cell RNA sequencing have delineated distinct subpopulations of fibroblasts in skin and other organs. To improve understanding of functional differences between fibroblast subpopulations, we have developed a novel technique to selectively isolate “migratory” fibroblasts, and non-migratory “resident” fibroblasts from heathy control (HC) and SSc skin. Objectives To compare migratory and resident populations of dermal fibroblasts in systemic sclerosis by their differential gene or protein expression, and through functional assays of fibrotic potential. Methods Forearm skin punch biopsies were collected from dcSSc(n=3), and healthy control skin(n=3). Migratory fibroblasts were first isolated by standard explant culture, then the residual biopsy fragments underwent collagenase digestion to yield a population of resident fibroblasts that retained in the biopsy fragments. These populations were further expanded for use at passage 3-6. Functional characterisation included 3-D collagen gel contraction, and migratory scratch-wound assays. Expression of pro-collagen I (Col1), CTGF and αSMA was compared by western blot. Bulk RNAseq on each fibroblast population was performed. Statistical analysis was carried out using Rsoftware “tidyverse”. Criteria for significant differences in gene expression were a fold change of ≥1.5, and adjusted p-value (FDR) of &lt;0.05 Results Compared with HC, all SSc fibroblast populations showed a hallmark fibrotic phenotype with increased gel contraction, faster migration, and overexpression of Col1, CTGF and αSMA compared with HC. However, SSc resident fibroblasts showed attenuated contraction, migration, and reduced levels of αSMA compared to migratory SSc fibroblasts. Bulk RNAseq performed on each fibroblast population confirmed 5579 significantly differentially expressed genes between SSc resident and SSc migratory fibroblasts, whereas no genes were significantly differentially expressed between HC resident and migratory fibroblasts. SSc and HC migratory fibroblasts and resident fibroblasts were then compared, to understand disease-related differences between the two fibroblast populations. 739 genes were significantly overexpressed in the migratory fibroblast population (including ASPM, TRIP3), whereas 745 genes were significantly upregulated in the resident fibroblast population. The genes upregulated in resident fibroblasts included CCL2, CXCL8, and ICAM1. Many genes typically associated with SSc (such as SERPINE1, COMP), were not significantly different between the SSc fibroblast subpopulations, but were significantly elevated in both SSc subpopulations compared to HC fibroblast populations, suggesting that these reflect a more generic SSc phenotype. Conclusion Migratory and resident SSc fibroblast populations exhibit distinct functional and transcriptional differences that are much less apparent for HC biopsies. Further work is required to understand the precise contribution these distinct SSc fibroblast populations in pathogenesis, and how they might be targeted therapeutically. Our findings also highlight that conventional explant culture techniques may ignore important fibroblasts populations and highlight the importance of more detailed analysis such as single cell analysis to better understand pathobiology of SSc. Table 1. Table of the most significantly differentially expressed genes with highest fold change and adjusted p-value (FDR) between the migratory and resident SSc fibroblast population Significantly upregulated genes in SSc migratory fibroblasts Significantly upregulated genes in SSc resident fibroblasts Fold change Adjusted p value Fold change Adjusted p value GPR1 5.99 0.01 CCL2 38.66 &lt;0.0001 PPME1 4.86 0.011 CXCL8 24.36 0.0001 KIF20A 4.34 0.015 ICAM1 15.38 &lt;0.0001 CENPF 4.31 0.018 EGR1 15.19 0.0002 STC2 4.18 0.01 EGR2 12.77 0.0002 ASPM 3.88 0.02 IFI44L 11.5 0.002 TRIB3 3.60 0.011 CXCL6 11.43 &lt;0.0001 HAPLN1 3.36 0.039 PDGF-D 10.14 0.0003 CCNB2 3.29 0.025 OAS3 8.94 0.0009 KIF14 3.24 0.027 IFIT1 8.58 0.004 Disclosure of Interests Kristina Clark: None declared, Alice Cole: None declared, Xu Shiwen: None declared, Voon Ong: None declared, Christopher D Buckley Employee of: founder of Mestag Therapeutics https://mestagtherapeutics.com/ , Christopher P Denton: None declared.